title: Introduction of a disulfide bond leads to stabilization and crystallization of a ricin immunogen.
abstract: RTA1-33/44-198 is a catalytically inactive, single-domain derivative of the ricin toxin A-chain (RTA) engineered to serve as a stable protein scaffold for presentation of native immunogenic epitopes (Olson et al., Protein Eng Des Sel 2004;17:391-397). To improve the stability and solubility of RTA1-33/44-198 further, we have undertaken the design challenge of introducing a disulfide (SS) bond. Nine pairs of residues were selected for placement of the SS-bond based on molecular dynamics simulation studies of the modeled single-domain chain. Disulfide formation at either of two positions (R48C/T77C or V49C/E99C) involving a specific surface loop (44-55) increased the protein melting temperature by ~5°C compared with RTA1-33/44-198 and by ~13°C compared with RTA. Prolonged stability studies of the R48C/T77C variant (> 60 days at 37°C, pH 7.4) confirmed a > 40% reduction in self-aggregation compared with RTA1-33/44-198 lacking the SS-bond. The R48C/T77C variant retained affinity for anti-RTA antibodies capable of neutralizing ricin toxin, including a monoclonal that recognizes a human B-cell epitope. Introduction of either R48C/T77C or V49C/E99C promoted crystallization of RTA1-33/44-198, and the X-ray structures of the variants were solved to 2.3 A or 2.1 A resolution, respectively. The structures confirm formation of an intramolecular SS-bond, and reveal a single-domain fold that is significantly reduced in volume compared with RTA. Loop 44 to 55 is partly disordered as predicted by simulations, and is positioned to form self-self interactions between symmetry-related molecules. We discuss the importance of RTA loop 34 to 55 as a nucleus for unfolding and aggregation, and draw conclusions for ongoing structure-based minimalist design of RTA-based immunogens.
authors.affiliation: Division of Biochemistry, Walter Reed Army Institute of Research, Silver Spring, Maryland 20910, USA.
authors.forename: Jaimee R
authors.initials: JR
authors.lastname: Compton
authors.forename: Patricia M
authors.initials: PM
authors.lastname: Legler
authors.forename: Benjamin V
authors.initials: BV
authors.lastname: Clingan
authors.forename: Mark A
authors.initials: MA
authors.lastname: Olson
authors.forename: Charles B
authors.initials: CB
authors.lastname: Millard
mesh_terms: D000818:Animals; D015153:Blotting, Western; D003460:Crystallization; D018360:Crystallography, X-Ray; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D051379:Mice; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D055503:Protein Multimerization; D055550:Protein Stability; D012276:Ricin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D044366:Transition Temperature
chemical_list: D004220:Disulfides; D000939:Epitopes; D007074:Immunoglobulin G; D012276:Ricin